This invention concerns a novel class of urea derivatives possessing aspartyl protease inhibitory properties. It describes the synthetic methodology used to make these urea derivatives from readily available L-lysine analogues and their biological applications. In addition, this invention relates to different pharmaceutical compositions comprising these compounds. The compounds and the pharmaceutical compositions of this invention have been shown to inhibit the activity of HIV aspartyl protease, an enzyme essential for virus maturation and infectivity. The inhibitory property may be advantageously used to provide compounds with antiviral properties against HIV viruses, including the HIV-1 and HIV-2 viruses.
HIV, the human immunodeficiency virus, causes AIDS through infection of specialized cells of the immune system carrying CD4 receptors. The HIV retrovirus reproduces in these cells, especially the so-called T-helper cells, and kills them in the process. While the body has the ability to re-generate T-helper cells to some extent, after years of continuous cell destruction by HIV and fighting back by the immune system, the virus eventually emerges as the battle""s winner. The progressive destruction of T-helper cells leads to weakening of the immune system which in turn, opens the door to opportunistic pathogens. When this happens, HIV-infected people start to show clinical symptoms. If left unchecked, HIV infection leads to death in a matter of years.
In order to reproduce in infected cells, HIV needs three major enzymes that are carried inside the viral particle. These three enzymes, reverse transcriptase, protease and integrase, thus represent ideal targets for antiviral therapy. Of these, reverse transcriptase has been the first enzyme targeted by the pharmaceutical industry. Inhibitors of the viral protease have been developed more recently and their use as drugs for AIDS treatment began only in 1996.
Although the development of reverse transcriptase and protease inhibitors has improved significantly the survival time and quality of life of HIV-infected patients, their use leads to unwanted side effects, such as anemia, neurotoxicity, bone marrow suppression and lipodystrophy. Most of the currently available anti-protease drugs are large molecules with limited ability to cross the blood-brain barrier. New compounds devoid of these drawbacks are urgently needed to treat HIV infections. In addition, HIV has the ability to develop resistance to the currently available drugs, so new compounds with original structure are desirable to fight these resistant viral strains.
The present invention provides a novel class of compounds, including their pharmaceutically acceptable derivatives. These compounds have an affinity for aspartyl proteases, in particular, HIV aspartyl protease. Therefore, these compounds are useful as inhibitors of such proteases. These compounds can be used alone or in combination with other therapeutic or prophylactic agents for the treatment or prophylaxis of viral infection.
According to a preferred embodiment, the compounds of this invention are capable of inhibiting HIV viral replication in human cells (e.g., CD4+ T-cells), by inhibiting the ability of HIV aspartyl protease to catalyse the hydrolysis of peptide bonds present in viral Gag and Gag-Pol polyproteins. These novel compounds can thus serve to reduce the production of infectious virions from acutely and chronically infected cells, and can inhibit the initial or further infection of host cells. Accordingly, these compounds are useful as therapeutic and prophylactic agents to treat or prevent infection by HIV-1 and HIV-2, which may result in asymptomatic infection, AIDS-related complex (ARC), acquired immunodeficiency syndrome (AIDS), AIDS-related dementia, or similar diseases of the immune system, and related viruses such as HTLV-I and HTLV-II, and simian immunodeficiency virus.
It is the main objective of this invention to provide a novel class of molecules that are aspartyl protease inhibitors, and particularly, HIV aspartyl protease inhibitors.
The present invention relates to a class of Nxcex5-amino acid substituted L-lysine derivatives (including its lower and higher homologues and analogs) as well as their pharmaceutically acceptable derivatives (e.g., salts).
Accordingly, the present invention in accordance with one aspect thereof provides a compound(s) of formula I 
and when the compound of formula I comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein n may be 3, 4 or 5, wherein Y may be O, S, NH or Nxe2x80x94CN,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOR5, and xe2x80x94CH2OR6 wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1 may be a benzenesulfonyl group of formula II 
wherein R2 may be selected from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, and a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R3 may be selected from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a piperonyl group, a phenyl group, a picolyl group selected from the group consisting of 
a thiophene group selected from the group consisting of 
and a benzyl group of formula III 
wherein R4 may be selected from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon, a piperonyl (i.e. 3,4-methylenedioxybenzyl) group, 1-indanyl, (R)-2-hydroxy-1-indanyl, (S)-2-hydroxy-1-indanyl, 1-isoquinolyl, 2-quinolyl, a group of formula IIIa 
a picolyl group selected from the group consisting of 
a thiophene group selected from the group consisting of 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of forrnula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
and a group of formula, 
wherein R5 may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms and a branched alkyl group of 3 or 4 carbon atoms,
wherein R6 may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms and a branched alkyl group of 3 or 4 carbon atoms,
wherein R7 and R8, same or different, may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
wherein R9 and R10, same or different, may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms and a branched alkyl group of 3 or 4 carbon atoms,
wherein m may be 0 or 1, wherein o may be 0, 1 or 2, and wherein p may be 0, 1 or 2.
In a further aspect, the present invention provides, a compound(s) of formula IA, 
and when the compound of formula IA comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein n may be 3, 4 or 5,
wherein Y may be O, S, NH or Nxe2x80x94CN,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOR5, and xe2x80x94CH2OR6 wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1 may be a benzenesulfonyl group of formula II 
wherein R2 may be selected from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, and a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
wherein R3 may be selected from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a piperonyl group, a phenyl group,
a picolyl group selected from the group consisting of 
a thiophene group selected from the group consisting of 
and a benzyl group of formula III 
wherein R4 may be selected from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon, a piperonyl (i.e. 3,4-methylenedioxybenzyl) group, 1-indanyl, (R)-2-hydroxy-1-indanyl, (S)-2-hydroxy-1-indanyl, 1-isoquinolyl, 2-quinolyl,
a group of formula IIIa 
a picolyl group selected from the group consisting of 
a thiophene group selected from the group consisting of 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of fonnula, 
a group of formula, 
a group of formula, 
a group of formula, 
a group of formula, 
and a group of formula, 
wherein R5 may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms and a branched alkyl group of 3 or 4 carbon atoms,
wherein R6 may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms and a branched alkyl group of 3 or 4 carbon atoms,
wherein R7 and R8, same or different, may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
wherein R9 and R10, same or different, may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, and a branched alkyl group of 3 or 4 carbon atoms,
wherein m may be 0 or 1, wherein o may be 0, 1 or 2, and wherein p may be 0, 1 or 2.
More particularly, the present invention provides compounds wherein Cx may be xe2x80x94CO2H, Y may be O and n may be 4.
In accordance with the present invention, R2 may be iso-butyl.
Also in accordance with the present invention, R1 may be selected from the group consisting of 4-CH3C6H4SO2xe2x80x94 and 4-NH2C6H4SO2xe2x80x94.
In accordance with the present invention, R1 may be selected from the group consisting of 4-CH3C6H4SO2xe2x80x94 and 4-NH2C6H4SO2xe2x80x94 while R2 may be iso-butyl.
The present invention also provides compounds wherein Cx may be xe2x80x94CH2OH, Y may be O and n may be 4.
In accordance with the present invention, R2 may be selected from the group consisting of iso-butyl, 2-methylbutyl and 3-methylbutyl.
Also in accordance with the present invention, R1 may be selected from the group consisting of 4-CH3C6H4SO2xe2x80x94 and 4-NH2C6H4SO2xe2x80x94 while R2 may be iso-butyl.
In accordance with the present invention, R1 may be 4-NH2C6H4SO2xe2x80x94 while R2 may be selected from the group consisting of 2-methylbutyl and 3-methylbutyl.
In addition, the present invention provides compounds wherein Cx may be xe2x80x94CO2H, Y may be S and n may be 4.
In accordance with the present invention, R1 may be 4-CH3C6H4SO2xe2x80x94 while R2 may be iso-butyl.
Furthermore, the present invention provides compounds wherein Cx may be xe2x80x94CH2OH, Y may be S and n may be 4.
In an additional aspect, the present invention provides a compound(s) of formula Ia 
and when the compound of formula Ia comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOH and xe2x80x94CH2OH, wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1a may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
and wherein R3, R4, R9 and R10 may be as defined above.
More particularly, the present invention provides compounds wherein R3 may be a group selected from the group consisting of 
Furthermore, the present invention provides compounds wherein R3 may be a group of formula IV 
where R3a may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH.
In yet a further aspect, the present invention provides a compound(s) of formula Ib 
and when the compound of formula Ib comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOH and xe2x80x94CH2OH, wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1b may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
and wherein R3, R4, R9 and R10 may be as defined above.
More particularly, the present invention provides compounds wherein Cx may be selected from the group consisting of xe2x80x94COOH and xe2x80x94COOM, and wherein M may be selected from the group consisting of Na, K and Cs.
In addition, the present invention provides compounds wherein Cx may be xe2x80x94CH2OH.
In another aspect, the present invention provide a compound(s) of formula Ic 
and when the compound of formula Ic comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOH and xe2x80x94CH2OH, wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1b may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
and wherein R3, R4, R9 and R10 may be as defined above.
More particularly, the present invention provides compounds wherein Cx may be xe2x80x94CH2OH.
In yet another aspect, the present invention provides compound(s) of formula Id 
and when the compound of formula Id comprises an amino group, pharmaceutically acceptable ammonium salts thereof,
wherein Cx may be selected from the group consisting of xe2x80x94COOM, xe2x80x94COOH and xe2x80x94CH2OH, wherein M may be selected from the group consisting of alkali metals (e.g., Na, K, Cs, etc) and alkaline earth metals (e.g., Ca, Mg, etc.),
wherein R1b may be selected from the group consisting of H, a straight alkyl group of 1 to 4 carbon atoms, a branched alkyl group of 3 or 4 carbon atoms, F, Cl, Br, I, xe2x80x94CF3, xe2x80x94NO2, xe2x80x94NR9R10, xe2x80x94NHCOR9, xe2x80x94OR9, xe2x80x94SR9, xe2x80x94COOR9, xe2x80x94COR9 and xe2x80x94CH2OH,
and wherein R3, R4, R9 and R10 may be as defined above.
More particularly, the present invention provides compounds wherein Cx may be xe2x80x94CH2OH.
It is to be understood herein that benzyl groups of formula III encompass, for example, without limitation, groups of formula 
It is also to be understood herein that groups of formula IIIa encompass for example, without limitation, groups of formula, 
This invention also provides in a further aspect, pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one compound of formula I, IA, Ia, Ib, Ic, and Id as defined herein. The pharmaceutical composition may comprise, for example, a pharmaceutically effective amount of such one or more compounds or as applicable, pharmaceutically acceptable ammonium salts thereof.
The term xe2x80x9cpharmaceutically effective amountxe2x80x9d refers to an amount effective in treating HIV infection in a patient. It is also to be understood herein that a xe2x80x9cpharmaceutically effective amountxe2x80x9d may be interpreted as an amount giving a desired therapeutic effect, either taken into one dose or in any dosage or route or taken alone or in combination with other therapeutic agents. In the case of the present invention, a xe2x80x9cpharmaceutically effective amountxe2x80x9d may be understood as an amount having an inhibitory effect on HIV (HIV-1 and HIV-2 as well as related viruses (e.g., HTLV-I and HTLV-II, and simian immunodeficiency virus)) infection cycle (e.g., inhibition of replication, reinfection, maturation, budding etc.) and on any organism depending on aspartyl proteases for their life cycle.
In addition, this invention provides pharmaceutical compositions in which these novel compounds of formula I, (as well as of formulae IA, Ia, Ib, Ic and Id) derived from L-lysine or L-lysine derivatives (as well as lower and higher homologues) are used to inhibit aspartyl proteases, including HIV aspartyl protease, thus providing protection against HIV infection.
The terms xe2x80x9cHIV proteasexe2x80x9d and xe2x80x9cHIV aspartyl proteasexe2x80x9d are used interchangeably and refer to the aspartyl protease encoded by the human immunodeficiency virus type 1 or 2. In a preferred embodiment of this invention, these terms refer to the human immunodeficiency virus type 1 aspartyl protease.
The term xe2x80x9cprophylactically effective amountxe2x80x9d refers to an amount effective in preventing HIV infection in a patient. As used herein, the term xe2x80x9cpatientxe2x80x9d refers to a mammal, including a human.
The terms xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d, xe2x80x9cpharmaceutically acceptable adjuvantxe2x80x9d and xe2x80x9cphysiologically acceptable vehiclexe2x80x9d refer to a non-toxic carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
The compounds of this invention include pharmaceutically acceptable derivatives of the compounds of formula I (as well as of formulae IA, Ia, Ib, Ic and Id) and as applicable pharmaceutically acceptable ammonium salts thereof. A xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an antivirally active metabolite or residue thereof.
It is to be understood herein that a xe2x80x9cstraight alkyl group of 1 to 6 carbon atomsxe2x80x9d includes for example, methyl, ethyl, propyl, butyl, pentyl, hexyl.
It is to be understood herein that a xe2x80x9cbranched alkyl group of 3 to 6 carbon atomsxe2x80x9d includes for example, without limitation, iso-butyl, tert-butyl, 2-pentyl, 3-pentyl, etc.
It is to be understood herein, that a xe2x80x9ccycloalkyl group having 3 to 6 carbonxe2x80x9d includes for example, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclocyclohexyl (i.e., C6H11).
Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and Nxe2x80x94(C1-4 alkyl)4+ salts.
The compounds of this invention contain one or more asymmetric carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomer, diastereomeric mixtures and individual diastereoisomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be of the R or S configuration.
Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term xe2x80x9cstablexe2x80x9d, as used herein, refers to compounds which possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art. Typically, such compounds are stable at a temperature of 40xc2x0 C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of such acid salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylhydrogensulfate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycollate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthylsulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, perchlorate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate.
This invention also envisions the quatemization of any basic nitrogen containing groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
It is to be understood herein, that if a xe2x80x9crangexe2x80x9d, xe2x80x9cgroup of substancesxe2x80x9d or particular characteristic (e.g., temperature, concentration, time and the like) is mentioned, the present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein. Thus, for example,
with respect to the number of carbon atoms, the mention of the range of 1 to 6 carbon atoms is to be understood herein as incorporating each and every individual number of carbon atoms as well as sub-ranges such as, for example, 1 carbon atoms, 3 carbon atoms, 4 to 6 carbon atoms, etc.
with respect to reaction time, a time of 1 minute or more is to be understood as specifically incorporating herein each and every individual time, as well as sub-range, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc.;
and similarly with respect to other parameters such as concentrations, elements, etc. . . .
It is in particular to be understood herein that the compound formulae each include each and every individual compound described thereby as well as each and every possible class or sub-group or sub-class of compounds whether such class or sub-class is defined as positively including particular compounds, as excluding particular compounds or a combination thereof.
It is also to be understood herein that xe2x80x9cgxe2x80x9d or xe2x80x9cgmxe2x80x9d is a reference to the gram weight unit and xe2x80x9cCxe2x80x9d, or xe2x80x9cxc2x0 C. xe2x80x9d is a reference to the Celsius temperature unit.
The compounds of this invention are easily prepared using conventional techniques from readily available and inexpensive starting materials. Two different strategies were used to prepared the new urea and thiourea derivatives. The first strategy uses an isocyanate (or thioisocyanate) intermediate obtained from a L-lysine derivative which is further reacted with diverse primary and secondary amines to yield the urea (or thiourea) end products. The second methodology uses a solid phase approach in which a L-lysine derivatives (or lysinol derivative) is transformed into various ureas upon reaction 1,1xe2x80x2-carbonyldiimidazole (or 1,1xe2x80x2-thiocarbonyldiimidazole) and a suitable amine synthon. The detailed description of these strategies are presented in schemes 1 to 6 discussed below.
Scheme 1 illustrates a generic example for the preparation of a key L-lysine intermediate IV needed for the synthesis of HIV protease inhibitors according to the first strategy (see schemes 1 and 2, see examples 2 and 18 in the experimental portion of this document).
Note:
a) For scheme 1, R1 represents a benzenesulfonyl group of formula II as defined above,
b) R2 represents an alkyl side chain as defined above (e.g., i-C4H9 (iso-butyl), 2-methylbutyl, 3-methylbutyl, etc.)
Scheme 1 can be used for the preparation of Nxcex1-isobutyl-Nxcex1-(4-methylbenzenesulfonyl)-L-lysine methyl ester (IVa) and Nxcex1-(4-aminobenzenesulfonyl)-Nxcex1-isobutyl-L-lysine methyl ester (IVb). It proceeds by using commercially available Nxcex5-benzyloxycarbonyl-L-lysine methyl ester hydrochloride (I) as the starting material. Reductive alkylation of derivative I with an appropriate aldehyde and sodium cyanoborohydride provided the derivative II. Then, sulfonation with benzenesulfonyl chloride (or substituted-benzenesulfonyl chloride) in the presence of triethylamine in dichloromethane gave compound III in excellent yields for the two first steps. Removal of the benzyloxycarbonyl group (Z group) by hydrogen gas in presence of 10% Pd/C yielded the free Nxcex5-amino derivative IV quantitatively. 
Scheme 2 illustrates a generic example for the preparation of HIV protease inhibitors bearing either a carboxylic function, compound VII, or an alcohol function, compound VIII, on the final product. In other words, this scheme shows the synthesis of a L-lysine urea derivative or a (2S) 2,6-diaminohexanol urea derivative.
Note:
a) For scheme 2, R1 and R2 are as defined in the first aspect of the invention,
b) R3 represents H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a picolyl group (i.e. 2-, 3-, or 4-picolyl), 2-thiophene-CH2xe2x80x94, 2-thiophene-CH2CH2xe2x80x94, a piperonyl group a phenyl or a benzyl group of formula III as defined above
c) R4 represents a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon, a group of formula IIIa as defined above, C5H10Nxe2x80x94CH2CH2xe2x80x94, OC4H8Nxe2x80x94CH2CH2xe2x80x94 (i.e. morpholine-4-CH2CH2xe2x80x94), (i.e. 2-picolyl), C5H4N-3-CH2xe2x80x94 (i.e. 3-picolyl), C5H4N-4-CH2xe2x80x94 (i.e. 4-picolyl), 2-thiophene-CH2xe2x80x94, 2-thiophene-CH2CH2xe2x80x94, piperonyl, 1-indanyl, (R)-2-hydroxy-1-indanyl, (S)-2-hydroxy-1-indanyl, indole-3-CH2CH2, C5H4N-2-CH2CH2xe2x80x94 (i.e. pyridine-2-ethyl), 1-isoquinol, 2-quinolyl, C6H5CH(CONH2), C6H5CH(CONH-i-C4H9), C6H5CH(CONHC6H5), 3-indole-CH2CH(CO2H), 2-HOxe2x80x94C6H4CONHxe2x80x94 or C5H4N-4-CONHxe2x80x94
Treatment of derivative IV with 1,1xe2x80x2-carbonyldiimidazole (or 1,1xe2x80x2-thiocarbonyldiimidazole) provided the intermediate isocyanate (or thioisocyanate) in excellent yields. This intermediate is stable in solution for months. The isocyanate (or thioisocyanate) can be treated with the desired primary or secondary amine to yield derivative VI in good to excellent yield. The final HIV protease inhibitors VII and VIII are easily obtained from the methyl ester VI by hydrolysis with sodium hydroxide in a mixture of THF and methanol giving the acid VII or by reduction with lithium aluminum hydride (LAH) giving the alcohol VIII, both in excellent yields. It is noteworthy that, under basic hydrolysis of VI to produce compound VII, some racemization may occur. However, it is not the case when compound VI is reduced with LAH to give derivative VIII. 
The derivatives VII (or VIII) can be further transformed into a variety of amidine or cyanoamidine of formula IX as shown in scheme 3. These transformations are done under standard reaction conditions. For example, the synthesis of cyanoamidine IX (Y=NCN) can be achieved upon reaction of VII with cyanamide in the presence of mercuric acetate for a period of 3 h. The amidine IX (Y=NH) derivatives is obtained as described above for the cyanamide by replacing cyanamide with ammonia.
Note:
a) For scheme 3, R represents Me or NH2,
b) Cx represents CO2H or CH2OH,
c) R3 and R4 are as defined above or in scheme 2
Scheme 4 illustrates a generic example for the transformation of L-lysine monohydrochloride into a Nxcex1,Nxcex1-disubstituted L-lysine derivatives such as, for example, Nxcex1-isobutyl-Nxcex1-(4-methylbenzenesulfonyl)-L-lysine hydrochloride.
Note:
a) For scheme 4, R1 represents a benzenesulfonyl group of formula II as defined in the first aspect of the invention,
b) R2 represents an alkyl side chain as defined above (e.g., i-C4H9 (iso-butyl), 2-methylbutyl, 3-methylbutyl, etc.)
As shown in scheme 4, the Nxcex1,Nxcex1-disubstituted L-lysine derivative XIV was obtained from commercially available L-lysine X in a four-step reaction sequence. This preparation uses the cyclic form of L-lysine in order to manipulate the Nxcex1-amino group without the need for protective groups. First, L-lysine was transformed into L-xcex1-amino-xcex5-caprolactam XI upon treatment with hydrochloric acid in methanol followed by neutralization with sodium hydroxide. The caprolactam XI is also commercially available. Reductive alkylation of derivative XI with an appropriate aldehyde and NaBH(OAc)3 in dichloroethane led to the Nxcex1-alkylamino-xcex5-caprolactam XII. Then, sulfonation with an arylsulfonyl chloride (or a substituted-arylsulfonyl chloride) in the presence of triethylamine in dichloromethane gave compound XIII in excellent yields. The Nxcex1,Nxcex1-disubstituted L-lysine derivative XIV was obtained quantitatively by acid hydrolysis of the cyclic amide XIII. 
Scheme 5 and 6 illustrate a second approach for the preparation of an anti-protease derivative using a solid phase methodology in accordance with the present invention. More specifically, scheme 5 presents the method for the synthesis of Nxcex1,Nxcex1-disubstituted-L-lysine derivatives (see example 1) and scheme 6 describes the synthesis of Nxcex1,Nxcex1-disubstituted-L-lysinol derivatives (see example 65). Any suitable solid phase substrate could be used in such preparation (K. Burgess, Solid phase organic synthesis, Wiley-Interscience, 2000).
Note:
a) For scheme 5, R1 is 4-methylbenzenesulfonyl, R2 is iso-butyl, R3 and R4 are as defined above.
This process allows the introduction of pharmacophores to a Nxcex1,Nxcex1-disubstituted-L-lysine derivative (such as XV) via the N-terminal function. Thus, in scheme 5, Nxcex1-isobutyl-Nxcex1-(4-methylbenzenesulfonyl)-Nxcex5-(9-fluorenylmethoxycarbonyl)-L-lysine XV is immobilized on a p-benzyloxybenzylalcohol resin (Wang resin) in DMF for a period of 16 h. The resulting component XVI contained 0.28 meq. of L-lysine derivative/g of resin. At this stage, after removal of the Fmoc protective group under standard reaction conditions (30% piperidine in DMF see T. W. Greene and P. G. M. Wuts, Protective groups in Organic Synthesis, 3rd Edition, John Wiley and Sons, Inc. 2000), the resin can be reacted with 1,1xe2x80x2-carbonyldiimidazole (or 1,1xe2x80x2-thiocarbonyldiimidazole) to give the intermediate isocyanate (or thioisocyanate) which is further treated with the an amine (or hydrazide) to yield component XVII. Cleavage of the resin with 95% trifluoroacetic acid (TFA) in CH2Cl2 leads to the desired L-lysine derivative VII. 
This second solid phase approach allows the introduction of pharmacophores to a Nxcex1,Nxcex1-disubstituted-L-lysinol derivative (such as XVIII) via the N-terminal function. Initially, (2S) 2-N-(4-aminobenzenesulfonyl)-2-N-isobutyl-6-N-(9-fluorenylmethoxycarbonyl)-2,6-diaminohexanol XVIII is immobilised on a trityl chloride resin in DCM for a period of 30 minutes. The resulting component IXX contained 0.19 meq. of L-lysinol derivative/g of resin. At this stage, after removal of the Fmoc protective group under standard reaction conditions, the resin can be reacted with 1,1xe2x80x2-carbonyldiimidazole (or 1,1xe2x80x2-thiocarbonyldiimidazole) to give the intermediate isocyanate (or thioisocyanate) which is then further treated with the an amine to yield component XX. Cleavage of the resin with, in this case, 1% trifluoroacetic acid (TFA) in CH2Cl2 for 3 h leads to the desired L-lysinol urea derivative VIII (i.e. (2S) 2,6-diaminohexanol urea). 
As it can be appreciated by the skilled artisan, the above synthetic schemes are not intended to be a comprehensive list of all means by which the compounds described and claimed in this application may be synthesized. Further methods will be evident to those of ordinary skill in the art.
The compounds of this invention may be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
As discussed above, the novel compounds of the present invention are excellent ligands for aspartyl proteases, particularly HIV-1 protease. Accordingly, these compounds are capable of targeting and inhibiting late stage events in the replication, i.e. the processing of the viral polyproteins by HIV encoded protease. Compounds according to this invention advantageously inhibit the ability of the HIV-1 virus to infect immortalized human T cells over a period of days, as determined by an assay measuring the amount of extracellular p24 antigenxe2x80x94a specific marker of viral replication (see, Meek et al., Nature, 343, pp. 90-92 (1990)).
In addition to their use in the prophylaxis or treatment of HIV or HTLV infection, the compounds according to this invention may also be used as inhibitory or interruptive agents for other viruses which depend on aspartyl proteases, similar to HW or HTLV aspartyl proteases, for obligatory events in their life cycle. Such compounds inhibit the proteolytic processing of viral polyprotein precursors by inhibiting aspartyl protease. Because aspartyl protease is essential for the production of mature virions, inhibition of that processing effectively blocks the spread of virus by inhibiting the production and reproduction of infectious virions, particularly from acutely and chronically infected cells. The compounds of this invention advantageously inhibit aspartyl proteases, thus blocking the ability of aspartyl proteases to catalyse the hydrolysis of peptide bonds.
The compounds of this invention may be employed in a conventional manner for the treatment or prevention of HIV, HTLV, and other viruses, which depend on aspartyl proteases for obligatory events in their life cycle. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques. For example, a compound of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a virally infected patient in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of the viral infection.
Alternatively, the compounds of this invention may be used in vaccines and methods for protecting individuals against viral infection over an extended period of time. The compounds may be employed in such vaccines either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of protease inhibitors in vaccines. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants conventionally employed in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against viral infections, such as HIV infection. As such, the novel protease inhibitors of this invention can be administered as agents for treating or preventing viral infections, including HIV infection, in a mammal.
The compounds of this invention may be administered to a healthy or HIV-infected patient either as a single agent or in combination with other antiviral agents which interfere with the replication cycle of HIV. By administering the compounds of this invention with other antiviral agents which target different events in the viral life cycle, the therapeutic effect of these compounds is potentiated. For instance, the co-administered antiviral agent can be one which targets early events in the viral life cycle, such as attachment to the cell receptor and cell entry, reverse transcription and viral DNA integration into cellular DNA. Antiviral agents targeting such early life cycle events include among others polysulfated polysaccharides, sT4 (soluble CD4) and other compounds which block binding of virus to CD4 receptors on CD4 bearing T-lymphocytes and other CD4(+) cells, or inhibit fusion of the viral envelope with the cytoplasmic membrane, and didanosine (ddI), zalcitabine (ddC), stavudine (d4T), zidovudine (AZT) and lamivudine (3TC) which inhibit reverse transcription. Other anti-retroviral and antiviral drugs may also be co-administered with the compounds of this invention to provide therapeutic treatment for substantially reducing or eliminating viral infectivity and the symptoms associated therewith. Examples of other antiviral agents include ganciclovir, dideoxycytidine, trisodium phosphonoformate, eflornithine, ribavirin, acyclovir, alpha interferon and trimenotrexate. Additionally, other types of drugs may be used to potentiate the effect of the compounds of this invention, such as viral uncoating inhibitors, inhibitors of Tat or Rev trans-activating proteins, antisense molecules or inhibitors of the viral integrase. These compounds may also be co-administered with other inhibitors of HIV aspartyl protease.
Combination therapies according to this invention exert a synergistic effect in inhibiting HIV replication because each component agent of the combination acts on a different site of HIV replication. The use of such combinations also advantageously reduces the dosage of a given conventional anti-retroviral agent that would be required for a desired therapeutic or prophylactic effect as compared to when that agent is administered as a monotherapy. These combinations may reduce or eliminate the side effects of conventional single anti-retroviral agent therapies while not interfering with the anti-retroviral activity of those agents. These combinations reduce the potential of resistance to single agent therapies, while minimizing any associated toxicity. These combinations may also increase the efficacy of the conventional agent without increasing the associated toxicity. Preferred combination therapies include the administration of a compound of this invention with AZT, 3TC, ddI, ddC, d4T or other reverse transcriptase inhibitors.
Alternatively, the compounds of this invention may also be co-administered with other HIV protease inhibitors such as Ro 31-8959 (Saquinavir; Roche), L-735,524 (Indinavir; Merck), AG-1343 (Nelfinavir; Agouron), A-84538 (Ritonavir; Abbott), ABT-378/r (Lopinavir; Abbott), and VX-478 (Amprenavir; Glaxo) to increase the effect of therapy or prophylaxis against various viral mutants or members of other HIV quasi species.
We prefer administering the compounds of this invention as single agents or in combination with retroviral reverse transcriptase inhibitors, or other HIV aspartyl protease inhibitors. We believe that the co-administration of the compounds of this invention with retroviral reverse transcriptase inhibitors or HIV aspartyl protease inhibitors may exert a substantial synergistic effect, thereby preventing, substantially reducing, or completely eliminating viral infectivity and its associated symptoms.
The compounds of this invention can also be administered in combination with immunomodulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyldithiocarbamate sodium, tumor necrosis factor, naltrexone and rEPO) antibiotics (e.g., pentamidine isethionate) or vaccines to prevent or combat infection and disease associated with HIV infection, such as AIDS and ARC.
When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this invention may be comprised of a combination of an aspartyl protease inhibitor of this invention and another therapeutic or prophylactic agent.
Although this invention focuses on the use of the compounds disclosed herein for preventing and treating HIV infection, the compounds of this invention can also be used as inhibitory agents for other viruses that depend on similar aspartyl proteases for obligatory events in their life cycle. These viruses include, but are not limited to, retroviruses causing AIDS-like diseases such as simian immunodeficiency viruses, HIV-2, HTLV-I and HTLV-II. In addition, the compounds of this invention may also be used to inhibit other aspartyl proteases and, in particular, other human aspartyl proteases including renin and aspartyl proteases that process endothelin precursors.
Pharmaceutical compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethyleneglycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of this invention may be administered orally, parenterally by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are amino acid, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv. or a similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspension and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene or polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable neat formulation. Topically-transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
Dosage levels of between about 0.01 and about 25 mg/kg body weight per day, preferably between about 0.5 and about 25 mg/kg body weight per day of the active ingredient compound are useful in the prevention and treatment of viral infection, including HIV infection. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the patient treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Preferably, such preparations contain from about 20% to about 80% active compound.
Upon improvement of a patient""s condition, a maintenance dose of a compound, composition or combination of this invention may be administered if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis, upon any recurrence of disease symptoms.
As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the infection, the patient""s disposition to the infection and the judgment of the treating physician.
The compounds of this invention are also useful as commercial reagents which effectively bind to aspartyl proteases, particularly HIV aspartyl protease. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide by an aspartyl protease, or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial aspartyl protease inhibitors will be evident to those of ordinary skill in the art.
In the description herein, the following abbreviations are used: